Test ID: PHIV    
HIV-1 Proviral DNA Qualitative Detection by PCR, Blood

HIV-1 Proviral DNA is extracted and purified from whole blood by the automated MagNA Pure LC instrument (Roche Applied Science, Indianapolis, IN) using the MagNA Pure LC DNA Isolation Kit-Large Volume. The specimen input volume is 500 mcL with a final elution volume of 110 mcL.

The double-stranded DNA is denatured by heat to expose the target region to labeled primers. The amplification target, a highly conserved region of the GAG genome, is bordered by the primer pair SK145 and SKCC1B. The 2 biotinylated oligonucleotide primers complementary to the amplification target sequence will bind to the target region on the DNA (native or internal control DNA). rTth polymerase links deoxynucleotide triphosphates, (dATP, dGTP, dCTP, dUTP) extending in the 5' to 3' direction to produce biotinylated complementary DNA sequences called amplicons. During PCR, controlled fluctuations in temperature allow repeated denaturation, annealing, and extension processes resulting in a geometric increase in the target sequences. DNA copies from previous cycles become templates in subsequent amplification periods. The resulting amplicon is 155 bp in length. HIV-1 internal control is added to identify clinical specimens containing inhibitory substances or test failure due to inadequate individual specimen processing. The internal control is introduced into each specimen during the extraction steps to serve as an extraction and amplification control for each independently processed specimen.

Uracil-N-glycosylase (UNG) is used to prevent contamination from previous PCR reactions. UNG will excise any dUTP found in previously amplified DNA. (Naturally occurring DNA will contain dTTP.) Before the PCR reaction is initiated, the thermal cycler is set at 55 degrees C to optimally activate UNG for 2 minutes. After the amplification cycles are completed, the UNG is inactivated by a denaturing solution containing sodium hydroxide.

The amplified DNA is incubated in polystyrene wells containing immobilized bovine serum albumin-conjugated probe (SK 102) that is specific to the biotinylated amplicons. After incubating for 1 hour, the unbound reactants are washed away. An avidin-horseradish peroxidase conjugate is added and incubated. Unbound reactants are washed away. A substrate solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) is added. In the presence of hydrogen peroxide, the bound horseradish peroxidase catalyzes the oxidation of TMB to form a color complex. The reaction is stopped by the addition of a weak acid and the absorbance is read at 450 nm. A fixed optical density cutoff is used to determine whether a specimen is positive or negative.(Package insert: Amplicor HIV-1 DNA Test, version 1.5, Roche Diagnostics, Indianapolis, IN, March 2005)

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